Báo cáo sinh học: " Carlow Virus, a 2002 GII.4 variant Norovirus strain from Ireland" pot

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Carlow Virus, a 2002 GII.4 variant Norovirus strain from Ireland
Karen Kearney*, John Menton and John G Morgan
Address: Lab 439, Department of Microbiology, University College Cork, College Road, Cork, Ireland
Email: Karen Kearney* - ; John Menton - ; John G Morgan -
* Corresponding author
Abstract
Background: Noroviruses are the leading cause of infectious non-bacterial gastroenteritis in
Ireland (population 4 million). Due to the number of outbreaks, its massive impact on the Irish
health service and its seasonality, Norovirus has gained public notoriety as The Winter Vomiting
Bug. The increase in cases in Ireland in the 2002–2003 season coincided with the emergence of two
new Genogroup II genotype 4 variant clusters of Norovirus worldwide.
Results: Little research has been done on the epidemiology or molecular biology of Norovirus
strains in Ireland. In an effort to combat this discrepancy, we cloned a full length human norovirus
genome as a cDNA clone (J3) which can produce full length transcripts in vitro. A polymerase
mutant cDNA clone (X1), in addition to a sub genomic cDNA clone (1A) were produced for use
in future work.
Carlow virus (Hu/NoV/GII/Carlow/2002/Ire) genome is 7559 nts in length, excluding the 3-end
poly A tail and represents the first Norovirus strain from Ireland to be sequenced.
Conclusion: Carlow virus is a member of the Farmington Hills variant cluster of Genogroup II
genotype 4 noroviruses.
Background
Noroviruses are the leading cause of infectious non-bacte-
rial gastroenteritis in Ireland.
The Department of Health, reported 7,500 cases of sus-
pected Norovirus infection in the 2002 season. The

National Disease Surveillance Centre stated that the
majority of these outbreaks occurred in a health care set-
ting with significant associated morbidity. Recent studies
are now implicating Norovirus as a cause of death, a fact
that has frequently been masked by the very location of
the outbreaks and their residents, namely nursing homes
and the elderly [1].
Norovirus is a member of the Caliciviridae family of
viruses. It is a single stranded, positive sense, RNA virus of
7.4–7.7 kb in length, with a 3' poly A tail. The genome is
organised into three Open Reading Frames (ORFs). ORF1
encodes an approximate 200 kDa polyprotein, which is
proteolytically processed into the N terminal protein,
NTPase, p22, p20, VpG, 3C-like protease and RNA
dependent RNA polymerase [2]. ORF2 and 3 encode the
structural proteins, capsid VP1 and minor structural pro-
tein VP2. The capsid is divided into two domains the N-
terminal shell (S) and the C-terminal protusion (P),
linked by an eight amino acid hinge. The P domain con-
sists of the P1 and P2 subdomains. The P2 domain located
on the surface of the capsid binds the histo-blood group
antigens [3,4].
Published: 13 June 2007
Virology Journal 2007, 4:61 doi:10.1186/1743-422X-4-61
Received: 10 April 2007
Accepted: 13 June 2007
This article is available from: />© 2007 Kearney et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( />),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Virology Journal 2007, 4:61 />Page 2 of 9

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Noroviruses are assorted into five Genogroups based on
sequence identity. Genogroup I, II and IV are associated
with human norovirus infection. Norovirus strains can be
further divided based on sequence homologies into 14 GI
and 17 GII genotypes [5]. GII.4 noroviruses are the pre-
dominant circulating genotype worldwide [6].
The increase in cases in Ireland in the 2002–2003 season,
coincided with the emergence of two new Genogroup II
genotype 4 variant clusters of Norovirus. These viruses
were part of a global epidemic that occurred in 1995 (US
95/96) and 2000 (Farmington Hills) [6]. These variant
viruses arise as a consequence of Norovirus being highly
adaptive, harbouring an RNA dependent RNA polymer-
ase, without proof reading capabilities, which allows a
high rate of mutations to accumulate, with many apparent
within the capsid receptor binding region. These muta-
tions may provide an opportunity to escape host immu-
nity [7,8,1].
Noroviruses are known to bind to the blood group anti-
gens in a strain specific manner [9]. The histo-blood
group antigens are highly polymorphic, a characteristic,
which may assist host immunity from infection, as these
complex carbohydrates also act as receptors for other
organisms such as H. pylori and E. coli R45 [10,11]. They
are present on the surfaces of red blood cells and on the
surface of the mucosal epithelium of the respiratory, gen-
itourinary and digestive tracts. They also exist as free oli-
gosaccharides, in biological fluids, such as saliva,
intestinal contents, milk and blood [[12-14] for review].

The heterogeneity of the blood group antigens in conjunc-
tion with the diversity of Norovirus strains has hampered
receptor binding studies due to the vast number of possi-
ble binding patterns. Advances have been made in the
study of human norovirus biology such as the develop-
ment of an in vitro replication system in a cell line [15-
17]. In addition, the propagation of murine norovirus in
cell culture has been a major accomplishment providing a
novel animal model [18].
Research on the epidemiology or molecular biology of
Norovirus strains in Ireland is at a very preliminary stage.
In an effort to overcome this we firstly screened 70 stool
samples from five different outbreaks that occurred in a
hospital setting to identify the most prevalent circulating
genotype. A representative of this genotype was then
cloned as a full length human norovirus genome, denoted
Carlow virus. Comparative sequence analysis was per-
formed using this virus against other noroviruses in the
database [GenBank] in order to identify differences, if
any, between Carlow virus and other viruses circulating
prior to the 2002 season. In addition, a polymerase
mutant genome and a representative clone of the sub
genomic RNA as cDNA clones were generated for use in
future work.
Results
Screening
70 stool samples were obtained from five different hospi-
tal outbreaks in the south eastern region of the country.
Screening of the 70 samples utilising COG2F [19] and the
reverse primer G2NVR designed to the 3' end of ORF1 and

start of ORF2 (Table 1) showed that 32 samples were pos-
itive for Norovirus. 28 were found to be members of
Genogroup II (GII), 2 of Genogroup I (GI) and 2 not yet
determined. The Genogroup I isolates belong to Genotype
1 and 3, respectively. Six of the 28 strains were sequenced
and were shown to be members of the Genogroup II gen-
otype 4 group of noroviruses. One such strain was cloned
in this study after only one round of RT-PCR, in conjunc-
tion with restriction endonuclease methodologies, as a
full length cDNA clone (J3) into pBluescript II SK+ and
was denoted Carlow virus.
The genome
The Carlow virus (Hu/NoV/GII/Carlow/2002/Ire)
genome is 7559 nts in length, excluding the 3' poly A tail
[GenBank: DQ415279
]. There are three ORFs, (ORF1, nt
5–5104; ORF2, nt 5085–6707; and ORF3, nt 6707–7513)
which harbour the potential to encode the non structural
polyprotein and the structural proteins, capsid VP1 and
minor structural protein, VP2. Blastn alignments of the
entire genome of Carlow virus revealed 99% identity to
the Norovirus GII genotype 4 variant members HU/NoV/
Farmington Hills/2002/USA [GenBank: AY502023
] and
the United Kingdom strain B4S6 [GenBank: AY587985
],
whereas only 90% identity was observed to the prototype
GII.4 member, Lordsdale virus [GenBank: X86557
].
Blastp alignments utilising the potential amino acid

sequences of all three ORFs were performed. The putative
polyprotein of Carlow virus (1700 a.a) exhibits 99% iden-
tity to a number of strains including HU/NoV/Farmington
Hills/2002/USA [GenBank: AY502023
], Hu/NoV/CS
G12002/USA [GenBank: AY502020.1
] and Hu/NLV/
Oxford/B4S2/2002/UK [GenBank: AAT00233.1
]. Carlow
virus showed 96% identity to the equivalent protein of
Lordsdale virus [GenBank: CAA60254
].
The potential capsid protein of Carlow virus conveys 99%
identity to a collection of strains such as Hu/NoV/Ger-
manton/2002/USA [GenBank: AAR97645.1
], Hu/NLV/
Oxford/B4S2/2002/UK [GenBank: AAT00234.1
], Hu/
NoV/Farmington Hills/2002/USA [GenBank:
AAR97663.1
] and NLV/GII/Langen1061/2002/DE [Gen-
bank: AAR32988.1
]. 93% identity was observed to the
capsid of Lordsdale virus [GenBank: CAA60255
].
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VP2 exhibits 98% to a variety of strains which includes
Hu/NLV/Oxford/B4S2/2002/UK [GenBank:
AAT00235.1

] and Hu/NoV/Farmington Hills/2002/USA
[GenBank: AY502023.1
]. 86% identity was observed to
VP2 of Lordsdale virus [GenBank: CAA60256
].
Transcription of the clones J3 (genomic), X1 (mutant)
and 1A (sub genomic) resulted in bands of approximately
7.5 kb for both J3 and X1, and a band of approximately
2.5 kb for 1A. An additional band of approximately 3.5 kb
was observed for the J3 and X1 reactions, which is pre-
sumably due to premature termination of transcription
(Figure 1).
Discussion
Carlow virus (Hu/NoV/GII/Carlow/2002/Ire) genome is
7559 nts in length, excluding the 3'-end poly A tail and
represents the first norovirus isolate from Ireland to be
sequenced. Alignments of the entire genome of Carlow
virus revealed 99% identity to the Norovirus GII Geno-
type 4 variant members HU/NoV/Farmington Hills/2002/
USA [GenBank: AY502023
] and the United Kingdom
strain B4S6 [GenBank: AY587985
]. Farmington Hills was
associated with 64% of all cruise ship outbreaks in the
United States, whereas its UK counterpart was implicated
in 100% of 22 outbreaks in Oxfordshire during 2002 and
2003 [20,8].
Genomes submitted prior to 2002 appeared to exhibit less
identity with Carlow virus such as the prototype GII.4
member Lordsdale virus [21] [GenBank: X86557

], which
displayed 90% identity over the entire genome. 'A novel
norovirus strain is defined as having 90% or less identity
at the nucleotide level with published sequences' [22]. In
addition, Carlow virus contains the six nucleotide motif
('AATCTG' at nt 4546–4552) not present in any geno-
group II genotype 4 noroviruses prior to 2002, and is
indicative of the norovirus variant genotype [23]. There-
fore, Carlow virus is a member of the Farmington Hills
variant cluster of Genogroup II genotype 4 noroviruses.
DNAstar EDITseq can predict the potential charge of a
protein. The most significant changes in this predicted
charge was for the polyprotein of Carlow virus with a
charge of 9.1 at pH 7 compared to Lordsdale virus, which
has a charge of 7.3 at pH 7. This change in charge is attrib-
utable to a number of amino acid changes between the
two proteins. Comparing proteins prior to and from
2002, a pattern of amino acids, which, appeared to be
conserved prior to 2002 were substituted for alternative
amino acids, which, appear to be conserved after 2002.
This phenomenon was evident for all three ORFs. Most of
these substitutions were with amino acids that were con-
served with regard to charge and/or structure. However,
for the majority of polyprotein sequences aligned, 6
amino acid substitutions, (before 2002) A, A, Q, Q, P, K
change to (after 2002) T, T, K, E, S and T, respectively (a.as
782, 791, 1045, 1291, 1456 and 1480) resulting in the
potential change in charge and/or structure of the poly-
protein.
In the majority of VP1 sequences, D

298
and N
394
were
replaced with N and G after 2002. These amino acids were
of interest as they were located within the predicted P2
domain of Carlow Virus. Based on sequence homologies,
the P2 domain of Carlow virus contains the NGR motif
(required for receptor binding) followed by a binding
pocket determined by Tan et al., 2003 [3]. This pocket
consists of an RGD-like motif (site I) and three additional
sites II, III and IV located between amino acids 268–418
(Figure 2). This pocket is considered to be responsible for
the binding of the human blood group antigens [3]. Nota-
bly, site IV mutates from DFQ (before 2002) to DFE (374–
377, after 2002) in a significant number of sequences
Table 1:
Primer Sequence Polarity Ref
JV12 ATACCACTATGATGCAGATTA (4279–4299) + [28]
SM31 CGATTTCATCATCACCATA (4592–4610) - [28]
COG2F CARGARBCNATGTTYAGRTGGATGAG (5003–5028) + [19]
G2NVR ACCNGCATANCCRTTRTACATTC (5365–5387) +
TX30SXN GACTAGTTCTAGATCGCGAGCGGCCGCCC (T × 30) - [29]
ORF3minusdegen ATCTCCTTRTCATGWTTRAARGAAGCC (6868–6894) -
430F ATGTGGGAYGGRGAGATCTAC (398–418) +
4440 minus TCGTTGATTGATATTGTGAAGTC (4436–4458) -
4440 nest TTGATTGATATTGTGAAGTC (4436–4455) -
5090 R TCATTCGACGCCATCTTCATT (5084–5104) -
4290 F TCACTATGATGCTGATTACTC (4282–4302) -
NLV1S25F GTGAATGAAGATGGCGTCTAACGAC (1–25) +

NEWRACE ATAGCAATTGTTGTCAAAGGCTGTGTAAGGGAACG (588–622) -
Virology Journal 2007, 4:61 />Page 4 of 9
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aligned (Figure 2). This mutation also occurred in Geno-
group II genotype 4 noroviruses in Japan between 1999
and 2004 [24].
Whether or not any of these amino acids have a role in
altering binding specificity would require mutational
analysis of the capsid protein, for use in, in vitro binding
assays.
Vp2 is a minor structural protein and has been shown to
stabilise the VP1 (VLP) particle and protect it from pro-
tease degradation in, in vitro studies [25]. In addition, it
has a highly basic charge, which has resulted in the sugges-
tion of a role in RNA binding [26]. The greatest degree of
divergence was observed for VP2 of Carlow virus when
comparing sequences in Genbank (98% to Farmington
Hills compared to 99% for the polyprotein and VP1 and
only 86% identity was observed to VP2 of Lordsdale virus
[GenBank: CAA60256
]). Interestingly, the amino acid
changes that occurred in VP2 of Carlow virus were prima-
rily conserved with regard to charge. One substitution, K
to E at amino acid 80 is of interest as it results in a signif-
icant change from a basic to an acidic residue. Mutational
analysis would be necessary to elucidate the impact of this
amino acid alteration.
Transcription of the clones J3 (genomic), X1 (mutant)
and 1A (sub genomic) resulted in bands of approximately
7.5 kb for both J3 and X1, and a band of approximately

2.5 kb for 1A. An additional band of approximately 3.5 kb
was observed for the J3 and X1 reactions, which is pre-
sumably due to premature termination of transcription.
Premature termination is observed for the transcription
reaction of the feline calicivirus vaccine strain 2024 from
the cDNA pIK12 clone [27].
Conclusion
In conclusion, Carlow virus is a member of the Geno-
group II, genotype 4 variant cluster of noroviruses, of
which Farmington Hills is the prototype.
Methods
Noroviruses
70 stool samples were obtained from five different sus-
pected Norovirus outbreaks from hospital settings in the
south eastern region of Ireland in the 2002–2003 season.
Random samples were taken from symptomatic individu-
als suspected to harbour Norovirus and stored at 4°C. Our
laboratory was notified to collect samples which were
then transported in sealed containers at 4°C.
RNA extraction and cDNA synthesis
Upon receipt of the samples, a 10% suspension was made
in DMEM (Sigma), vortexed briefly and clarified by cen-
trifugation at 10,000 rpm for 10 min. RNA was extracted
from 140 μl of supernatant using the Viral RNA mini kit
(Qiagen) and eluted in 50 μl of AVE buffer according to
the manufacturers instructions. The RNA was treated with
2 μl DNase I (Ambion) and stored at -80°C.
Standard Reverse Transcription
Reverse transcription was performed with 5 μl of RNA
template utilising SuperScript ™ II reverse transcriptase

(Invitrogen) and random hexamer primers (Roche) as
outlined in the manufacturers instructions.
Assembling the full length clone by Polymerase Chain
Reaction (PCR)
5 μl of sample 13 cDNA was used as a template for ampli-
fication in PCR. Primers JV12 and SM31 [28], which are
specific for the polymerase gene of Norovirus were used to
amplify a 333 nt fragment. This fragment was cloned into
the Topo II dual vector from Invitrogen, transformed into
Top10 cells (Invitrogen) in the presence of Ampicillin
(Sigma) 100 μg/ml, sequenced (MWG Biotech) and was
designated Pol9.
Screening
Screening primers COG2F (Table 1, [19]) and a reverse
primer G2NVR designed in our laboratory, (Table 1) were
used to generate a 384 nt PCR product from the ORF1/
(a) Denaturing agarose gel electrophoresis, of the RNA tran-scription products from J3 cDNAFigure 1
(a) Denaturing agarose gel electrophoresis, of the RNA tran-
scription products from J3 cDNA. Lane 1. 5 ul of J3 uncapped
transcript. Lane 2. 10 ul of J3 uncapped transcript. Lane 3. 1
kb NEB RNA ladder. (b) In vitro Transcription. Lane 1. J3
uncapped transcript. Lane 2. X1S mutant uncapped tran-
script. Lane 3. 1A sub genomic uncapped transcript. Lane 4.
1A sub genomic uncapped transcript.
(a) Lane 1 2 3
Bases
-9,000
-7,000
-5,000
-3,000

-2000
-1000
-500
(b)
Virology Journal 2007, 4:61 />Page 5 of 9
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The alignment of amino acids 241–419 and 481–540 of the potential capsid protein of Carlow virus, with the corresponding proteins of 12 post and 11 pre 2002 virusesFigure 2
The alignment of amino acids 241–419 and 481–540 of the potential capsid protein of Carlow virus, with the corresponding
proteins of 12 post and 11 pre 2002 viruses. The red circle indicates amino acids conserved prior to 2002 which have been
substituted for an amino which are conserved in the majority of viruses after 2002 (exception Lanzhou/35666/2002/china and
Yuri 32073/Japan). These changes are to un-conserved amino acids, acidic to basic for example. The thick black line shows the
RGD motif and 3 additional motifs responsible for strain specific binding of the human blood group antigens [3].
ABD73936 241 RFPIPLEKLFTGPSGAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHIAGTHNYT 300
AAR97645 241 300
AAT00234 241 A 300
AAT00327 241 300
AAR97663 241 300
AAS89040 241 300
AAT00339 241 300
AAT00249 241 A 300
AAR97651 241 S 300
AAT00348 241 300
AAR32988 241 300
ABC96746 241 S D 300
BAF38397 241 Y S A L S.D 300
AAL18873 241 Y S A T.S.D 300
AAD40497 241 Y S A S.D 300
AAT12693 241 Y S A S.D 300
AAD40488 241 Y S A S.D 300
AAT12696 241 Y S A S.D 300

AAL79839 241 Y S A S.D 300
AAL18876 241 Y S I A S.D 300
AAT12689 241 Y S A S.D 300
AAT12681 241 Y S A S.D 300
AAT12682 241 Y S A S.D 300
CAE47529 241 N D 300
ABD73936 301 MNLASQNWNNYDPTEEIPAPLGTPDFVGRIQGMLTQTTRGDGSTRGHKATVSTGDVHFTP 360
AAR97645 301 360
AAT00234 301 360
AAT00327 301 360
AAR97663 301 360
AAS89040 301 360
AAT00339 301 360
AAT00249 301 360
AAR97651 301 360
AAT00348 301 360
AAR32988 301 G 360
ABC96746 301 K V S 360
BAF38397 301 K S A S 360
AAL18873 301 K S 360
AAD40497 301 K A S 360
AAT12693 301 K I E A S 360
AAD40488 301 K E A S 360
AAT12696 301 K E A S 360
AAL79839 301 K E A S 360
AAL18876 301 K E A S 360
AAT12689 301 K E A S 360
AAT12681 301 K E A S 360
AAT12682 301 K E A S 360
CAE47529 301 K V.S S 360

ABD73936 361 KLGSIQFNTDTNNDFETGQNTKFTPVGVVQDGNGTHQNEPQQWVLPSYSGRTGHNVHLAP 420
AAR97645 361 420
AAT00234 361 420
AAT00327 361 420
AAR97663 361 420
AAS89040 361 R 420
AAT00339 361 V A 420
AAT00249 361 420
AAR97651 361 420
AAT00348 361 R 420
AAR32988 361 420
ABC96746 361 V T S N 420
BAF38397 361 V.YT I N N 419
AAL18873 361 T I SSA N.L P 420
AAD40497 361 V T Q T D-N N 419
AAT12693 361 V.YT Q I N 419
AAD40488 361 V.YT Q I N N 419
AAT12696 361 V.YT Q I N 419
AAL79839 361 V.YT Q I N N 419
AAL18876 361 V.YT Q I N N 419
AAT12689 361 V.YT Q I N N 419
AAT12681 361 V.YT Q I N N 419
AAT12682 361 V.YT Q I N N 419
CAE47529 361 V.YT L.A S N N V. 419
ABD73936 481 DTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPMGNGTGRRRAL 540
AAR97645 481 540
AAT00234 481 540
AAT00327 481 540
AAR97663 481 540
AAS89040 481 540

AAT00339 481 540
AAT00249 481 T 540
AAR97651 481 540
AAT00348 481 540
AAR32988 481 540
ABC96746 481 A 540
BAF38397 480 P A 539
AAL18873 481 Y P V 540
AAD40497 480 P A 539
AAT12693 480 P A 539
AAD40488 480 P A 539
AAT12696 480 P A 539
AAL79839 480 P A 539
AAL18876 480 P A 539
AAT12689 480 P A 539
AAT12681 480 P A 539
AAT12682 480 P A 539
CAE47529 480 P 539
Carlow/2002/Ire
Germanton/2002/USA
Oxford/B4S2/2002/UK
Oxford/B5S7/2002/UK
Farmington Hills/2002/USA
Oxford/B5S22/2003
Oxford/B5S13/2002/UK
Oxford/B4S1/2002/UK
Anchorage/2002/USA
Oxford/B6S6/2002/UK
Langen1061/2002/DE
Lanzhou/35666/2002/CHINA

YURI 32073
Erfurt/007/00/DE
408/97003012/1996/FL
Hamburg048/1997/GE
345/96002726/1996/SC
Hamburg139/1997/GE
DIJON171/96
Beeskow/124/00/DE
Bochum136/1998/GE
Dresden153/1997/GE
Dresden245/1997/GE
Ast6139/01/Sp
Carlow/2002/Ire
Germanton/2002/USA
Oxford/B4S2/2002/UK
Oxford/B5S7/2002/UK
Farmington Hills/2002/USA
Oxford/B5S22/2003
Oxford/B5S13/2002/UK
Oxford/B4S1/2002/UK
Anchorage/2002/USA
Oxford/B6S6/2002/UK
Langen1061/2002/DE
Lanzhou/35666/2002/CHINA
YURI 32073
Erfurt/007/00/DE
408/97003012/1996/FL
Hamburg048/1997/GE
345/96002726/1996/SC
Hamburg139/1997/GE

DIJON171/96
Beeskow/124/00/DE
Bochum136/1998/GE
Dresden153/1997/GE
Dresden245/1997/GE
Ast6139/01/Sp
Carlow/2002/Ire
Germanton/2002/USA
Oxford/B4S2/2002/UK
Oxford/B5S7/2002/UK
Farmington Hills/2002/USA
Oxford/B5S22/2003
Oxford/B5S13/2002/UK
Oxford/B4S1/2002/UK
Anchorage/2002/USA
Oxford/B6S6/2002/UK
Langen1061/2002/DE
Lanzhou/35666/2002/CHINA
YURI 32073
Erfurt/007/00/DE
408/97003012/1996/FL
Hamburg048/1997/GE
345/96002726/1996/SC
Hamburg139/1997/GE
DIJON171/96
Beeskow/124/00/DE
Bochum136/1998/GE
Dresden153/1997/GE
Dresden245/1997/GE
Ast6139/01/Sp

Carlow/2002/Ire
Germanton/2002/USA
Oxford/B4S2/2002/UK
Oxford/B5S7/2002/UK
Farmington Hills/2002/USA
Oxford/B5S22/2003
Oxford/B5S13/2002/UK
Oxford/B4S1/2002/UK
Anchorage/2002/USA
Oxford/B6S6/2002/UK
Langen1061/2002/DE
Lanzhou/35666/2002/CHINA
YURI 32073
Erfurt/007/00/DE
408/97003012/1996/FL
Hamburg048/1997/GE
345/96002726/1996/SC
Hamburg139/1997/GE
DIJON171/96
Beeskow/124/00/DE
Bochum136/1998/GE
Dresden153/1997/GE
Dresden245/1997/GE
Ast6139/01/Sp
Virology Journal 2007, 4:61 />Page 6 of 9
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A schematic representation of the genomic organisation of Carlow virusFigure 3
A schematic representation of the genomic organisation of Carlow virus. ORF's 1, 2 and 3 are indicated by arrows. Restriction
sites and the clones necessary for generation of the genome are shown. Thickened lines indicate vector sequences whereas
arrows represent the T3 and T7 phage promoters.

nt 4283-5104
15b
T3
nt 1-7559
J3
nt 1-5104
610
nt 398-5104
P3
nt 4338-7559
710
nt 398-4458
Poly6
nt 1-622
5p6
nt 5003-7559
1A
533 nt 4338 nt 5034nt
Bst11071 XbaI BglII
nt 1 nt 7559
nt 7559
XbaI BglII
Bst11071 XbaI BglII
Bst11071
XbaI BglII
XbaI BglII
BglII
533 nt 4338 nt 5034nt
Bst11071 XbaI BglII
nt 1

ORF1 ORF2 ORF3
Bst11071
NotI SalI
T7
Virology Journal 2007, 4:61 />Page 7 of 9
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ORF2 overlap region. 28 from 70 stool samples obtained
from 2002 generated a product utilising these primers. Six
of these sample products were sequenced (MWG Biotech)
and found to be Genogroup II genotype 4. One such sam-
ple product (sample 13) was cloned into pCR2.1 vector
(Invitrogen) to generate caps2 and 5 clones.
To generate the 3' half of the genome, the reverse tran-
scription reaction was performed utlising 10 μl of sample
13 RNA, 50 pmoles of TX30SXN [29] and 20 mM dNTPS
according to the manufacturers instructions using 1 ul of
Thermoscript (Invitrogen). PCR was employed using
primers ORF3minusdegerate (Table 1) and COG2F (Table
1, [19]) in a 50 μl volume utilising 1 μl of Platinum
®
Taq
DNA Polymerase High Fidelity (Invitrogen) as outlined
by the manufacturer.
A 2.9 kb fragment was cloned into the EcoRV (Roche)
digested pBlueScript II SK+ vector (Stratagene). The subse-
quent clone assigned 1A contained nts 5003 to 7559 of
Carlow Virus (Figure 3). This band was as a result of carry
over of the excess TX30SXN [29] utilised in the reverse
transcription and COG2F primer [19].
To generate the 5' half of the genome, reverse transcrip-

tion was approached as for the 3' end with the following
exceptions; 5 μl of sample 13 RNA and 50 pmoles of 4440
minus (Table 1) were utilised. PCR was performed using
5 μl of sample 13 cDNA, 50 pmoles of primers 430F and
4440 nest (Table 1) as previously described with the inclu-
sion of a final concentration of 1% Dimethylsulfoxide
(DMSO) (Sigma).
The resultant 4 kb band was cloned into the EcoRV
(Roche) digested pBlueScript II SK+ vector. The subse-
quent clone designated Poly6 harboured nt 398 to 4458
of Carlow Virus (Figure 3).
In order to fill the gap between nt 4458 to 5003, 2 μl of
the cDNA generated from the 3' end transcription, was
utilised as a template in addition to 50 pmoles of 5090
reverse, 4290 forward primers (Table 1) and 1 μl Plati-
num
®
Taq DNA Polymerase High Fidelity in a PCR reac-
tion according to the manufacturers instructions. The 827
nt fragment was cloned into the EcoRV (Roche) digested
pBlueScript II SK+ and labelled 15b. This clone contains
nt 4283 to 5104 of Carlow virus (Figure 3).
The immediate 5' end of Carlow Virus was determined
utilising cDNA generated from a standard reverse tran-
scription reaction, utilising random hexamer primers. The
PCR reaction utilised 1 μl of 50 pmol/μl of NLV1S25,
NEW RACE primers, (Table 1) and Taq polymerase
(FINNZYME Oy) as directed by the manufacturer. A 622
nt fragment was generated and cloned and assigned 5p6.
Nucleotide (nt) 1 – 24 corresponds to the primer, nt 25 –

622 corresponds to Carlow virus (Figure 3) (note: Numer-
ous attempts at 5' RACE (Invitrogen) to generate the
immediate 5' end did not yield sequence between nt 1 and
36).
The purpose of the following cloning experiments was to
link the four clones 5p6, Poly6, 15b and 1A by employing
restriction enzymes rather than subjecting the template to
any additional rounds of PCR.
The 819 nucleotide fragment of 15b was excised with XbaI
(Roche) and cloned into XbaI digested Poly6 to yield
pBlueScript II SK+ (Stratagene) harbouring nt 398–5104
of Carlow virus. This construct was sequenced to ensure
correct alignment, and was designated P3 (Figure 3, nt
398–5104 of Carlow virus).
710 is the 696 nt XbaI-BglII (nt 4338–5034) fragment of
15b cloned into the 5.8 kb BglII-XbaI (Roche) 1A fragment
and was confirmed by sequencing (Figure 3, nt 4338–
7559 of Carlow virus).
Clone 610 is the 639 nt XhoI-Bst11071 (Roche) fragment
of 5p6 cloned into the SalI-Bst11071 (Roche) digested P3
clone and confirmed by sequencing (Figure 3, nt 1–5104
of Carlow virus).
The 4.3 kb NotI-XbaI (Roche) fragment of 610 (nt 1–
4338) was cloned into the 6.3 kb XbaI -NotI (nt 4338–
7559) fragment of 710, to yield J3. This clone was
sequenced to ensure the correct alignment of the entire
7759 nucleotide Carlow virus genome (Figure 3, nt 1–
7559).
The polymerase mutant genome denoted X1
X1 was generated by XbaI digestion of J3, followed by end

filling using Klenow enzyme (Promega) in the presence of
1.1 μM dNTPs as outlined by the manufacturer. Sequence
analysis confirmed the insertion of the four nucleotides
CTAG within the polymerase gene at nt 4346 of Carlow
virus [GenBank: DQ415279
]. This mutation results in a
stop codon in the predicted amino acid sequence of the
Carlow virus non structural polyprotein at amino acid
1454.
In vitro transcriptions
pBlueScript II SK+ (Stratagene) possesses both a T3 and T7
phage promoter either side of the multiple cloning region.
The clones J3, X1 and 1A all have a T3 promoter upstream
of the cloned viral cDNA and a T7 promoter downstream.
The poly A tail is followed by a SalI restriction site (Figure
3). The 5' end is preceded by an NotI site (Figure 3).
Virology Journal 2007, 4:61 />Page 8 of 9
(page number not for citation purposes)
Capped and uncapped RNA transcripts were produced uti-
lising 1 μg of SalI digested J3, X1 and 1A cDNAs respec-
tively, in standard in vitro transcription reactions, in a 120
μl volume, utilising the Ambion Megascript
®
T3 kit
(Ambion) and manual. The samples were incubated at
37°C for 6 hours, treated with Dnase I (Ambion) for 1
hour at 37°C. The resultant transcripts were treated with
3 μl of 0.5 M EDTA and cleaned using a High Pure PCR
Purification Kit (Roche). The RNA was resuspended in 50
μl of RNase free, DNase free H

2
O. The concentration of
plus strand RNA was determined by spectrophotometry.
The quality of RNA was determined by denaturing gel
electrophoresis.
Competing interests
Department of Microbiology, University College Cork
Irish Government
Authors' contributions
Dr. Karen Kearney is the corresponding and main contrib-
uting author of this manuscript. Screening results were
provided by John Menton. Supervision and final review
was contributed by Dr. John Morgan. All authors have
read and approved this manuscript.
Acknowledgements
The project was funded by the Irish Government under the National Devel-
opment Plan 2000–2006. We greatly appreciate the contribution of the
staff at the Waterford Regional Hospital Medical Laboratory. In addition,
we would like to thank Maurice O'Donoghue, Liam Burgess and Siobhan
Cashman for technical assistance. We would like to acknowledge the assist-
ance of Dr Kim Green and Dr Stanislav Sosnovtsev at the National Insti-
tutes for Allergy and Infectious Disease, Bethesda, Maryland, USA.
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